Papaya ringspot virus protease gene

ABSTRACT

NIa protease genes of papaya ringspot virus strains FLA.83 W and USA P-type (HA attenuated) strain are provided.

FIELD OF THE INVENTION

This invention relates to a protease gene derived from papaya ringspotvirus. More specifically, the invention relates to the geneticengineering of plants and to a method for conferring viral resistance toa plant using an expression cassette encoding papaya ringspot virus PRVFLA.83 W or PRV USA P-type (HA attenuated) protease.

BACKGROUND OF THE INVENTION

Many agriculturally important crops are susceptible to infection byplant viruses, particularly papaya ringspot virus, which can seriouslydamage a crop, reduce its economic value to the grower, and increase itscost to the consumer. Attempts to control or prevent infection of a cropby a plant virus such as papaya ringspot virus have been made, yet viralpathogens continue to be a significant problem in agriculture.

Scientists have recently developed means to produce virus resistantplants using genetic engineering techniques. Such an approach isadvantageous in that the genetic material which provides the protectionis incorporated into the genome of the plant itself and can be passed onto its progeny. A host plant is resistant if it possesses the ability tosuppress or retard the multiplication of a virus, or the development ofpathogenic symptoms. "Resistant" is the opposite of "susceptible," andmay be divided into: (1) high, (2) moderate, or (3) low resistance,depending upon its effectiveness. Essentially, a resistant plant showsreduced or no symptom expression, and virus multiplication within it isreduced or negligible. Several different types of host resistance toviruses are recognized. The host may be resistant to: (1) establishmentof infection, (2) virus multiplication, or (3) viral movement.

Potyviruses are a distinct group of plant viruses which are pathogenicto various crops, and which demonstrate cross-infectivity between plantmembers of different families. Generally, a potyvirus is asingle-stranded RNA virus that is surrounded by a repeating proteinmonomer, which is termed the coat protein (CP). The majority of thepotyviruses are transmitted in a nonpersistent manner by aphids. As canbe seen from the wide range of crops affected by potyviruses, the hostrange includes such diverse families of plants as Solanaceae,Chenopodiaceae, Gramineae, Compositae, Leguminosae, Dioscroeaceae,Cucurbitaceae, and Caricaceae. Potyviruses include watermelon mosaicvirus II (WMVII); zucchini yellow mosaic virus (ZYMV), potato virus Y,tobacco etch and many others.

Another potyvirus of economic significance is papaya ringspot virus(PRV). Two groups of PRV have been identified: the "P" or "papayaringspot" type infects papayas; and the "W" or "watermelon" type infectscucurbits, e.g., squash, but it is unable to infect papaya. Thus, thesetwo groups can be distinguished by host range differences.

The potyviruses consist of flexous, filamentous particles of dimensionsapproximately 780×12 nanometers. The viral particles contain asingle-stranded positive polarity RNA genome containing about 10,000nucleotides. Translation of the RNA genome of potyviruses shows that theRNA encodes a single large polyprotein of about 330 kD. This polyproteincontains several proteins; these include the coat protein, nuclearinclusion proteins NIa and NIb, cytoplasmic inclusion protein (CI), andother proteases and movement proteins (see FIG. 1). These proteins arefound in the infected plant cell and form the necessary components forviral replication. One of the proteins contained in the polyprotein is a35 kD capsid or coat protein which coats and protects the viral RNA fromdegradation. One of the nuclear inclusion proteins, NIb, is an RNAreplicase component and is thought to have polymerase activity. CI, asecond inclusion protein, is believed to participate in the replicasecomplex and have a helicase activity. NIa, a third inclusion protein,has a protease activity. In the course of potyvirus infection, NIa andNIb are translationally transported across the nuclear membrane into thenucleus of the infected plant cell at the later stages of infection andaccumulate to high levels.

The location of the protease gene appears to be conserved in theseviruses. In the tobacco etch virus, the protease cleavage site has beendetermined to be the dipeptide Gln-Ser, Gln-Gly, or Gln-Ala.Conservation of these dipeptides at the cleavage sites in these viralpolyproteins is apparent from the sequences of the above-listedpotyviruses.

Expression of the coat protein genes from tobacco mosaic virus, alfalfamosaic virus, cucumber mosaic virus, and potato virus X, among others,in transgenic plants has resulted in plants which are resistant toinfection by the respective virus. For reviews, see Fitchen et al.,Annu. Rev. Microbiol., 4, 739 (1993) and Wilson, Proc. Natl. Acad. Sci.USA, 90 3134 (1993). For papaya ringspot virus, Ling et al.(Bio/Technology, 9, 752 (1991)) found that transgenic tobacco plantsexpressing the PRV coat protein (CP) gene isolated from the PRV strainHA 5-1 (mild) showed delayed symptom development and attenuation ofsymptoms after infection by a number of potyviruses, including tobaccoetch (TEV), potato virus Y (PVY), and pepper mottle virus (PeMV). PRVdoes not infect tobacco, however. Thus, PRV CP transgenic tobacco plantscannot be used to evaluate protection against PRV. Fitch et al.(Bio/Technology, 10 , 1466 (1992)), Gonsalves (American J. of Bot., 79,88 (1992)), and Lius et al. (91st Annual Meeting of the American Societyfor Horticultural Science Hortscience, 29, 483 (1994)) reported thatfour R_(o) papaya plants made transgenic for a PRV coat protein genetaken from strain HA 5-1 (mild) displayed varying degrees of resistanceagainst PRV infection, and one line (S55-1) appeared completelyresistant to PRV. This appears to be the only papaya line that showscomplete resistance to PRV infection.

Even though coat protein mediated viral resistance has proven to beuseful in a variety of situations, it may not always be the mosteffective or desirable means for providing viral resistance. In suchinstances, it would be advantageous to have other methods for conferringviral resistance to plants. Expression of the protease gene (NIa) fromtobacco vein mottle virus (TVMV) and potato virus Y (PVY) in transgenicplants has shown the feasibility of using protease gene constructs toproduce transgenic plants protected against potyvirus infection (Maitiet al., J. Cell. Biochem., Suppl. 16F, 217(1992); Vardi et al., Proc.Natl. Acad. Sci. U.S.A., 90, 7513 (1993); Maiti et al., Proc. Natl.Acad. Sci. U.S.A. 90, 6110 (1993)). Maiti et al. (1993) showed that theexpression of the NIa gene of TVMV in tobacco plants rendered theseplants highly resistant to TVMV challenge. In addition, Maiti et al.showed that the NIa gene expressed in these plants was proteolyticallyactive. Vardi et al. transformed tobacco plants with PVY NIa constructs.R₁ progeny from two lines derived from these transformed plants wereresistant to challenge with virus.

There is a continuing need for the transgenic expression of genesderived from potyviruses at levels which confer resistance to infectionby these viruses.

SUMMARY OF THE INVENTION

This invention provides an isolated and purified DNA molecule thatencodes the protease for the FLA83 W-type strain of papaya ringspotvirus (PRV) or the protease for the PRV USA P-type (HA attenuated)strain. This invention also provides an isolated and purified DNAmolecule that encodes the protease and flanking gene segments for theFLA83 W-type strain of papaya ringspot virus (PRV) or the protease andflanking gene segments for the PRV USA P-type (HA attenuated) strain.The invention also provides a chimeric expression cassette comprising atleast one of these DNA molecules, a promoter which functions in plantcells to cause the production of an RNA molecule, and at least onepolyadenylation signal comprising 3' nontranslated DNA which functionsin plant cells to cause the termination of transcription and theaddition of polyadenylated ribonucleotides to the 3' end of thetranscribed mRNA sequences, wherein the promoter is operably linked tothe DNA molecule, and the DNA molecule is operably linked to thepolyadenylation signal. Another embodiment of the invention isexemplified by the insertion of multiple virus gene expression cassettesinto one purified DNA molecule, e.g., a plasmid. Preferably, thesecassettes include the promoter of the 35S gene of cauliflower mosaicvirus and the polyadenylation signal of the cauliflower mosaic virus 35Sgene.

Also provided are bacterial cells, and transformed plant cells,containing the chimeric expression cassettes comprising the proteasegene derived from the FLA.83 W-type strain of papaya ringspot virus(referred to herein as PRV FLA83 W) or from the USA P-type (HAattenuated) strain of PRV, and preferably the 35S promoter ofcauliflower mosaic virus and the polyadenylation signal of thecauliflower mosaic virus 35S gene. Plants are also provided, wherein theplants comprise a plurality of transformed cells transformed with anexpression cassette containing the protease gene derived from the PRVFLA83 W strain or from the USA P-type (HA attenuated) strain of PRV, andpreferably the cauliflower mosaic virus 35S promoter and thepolyadenylation signal of the cauliflower mosaic virus gene. Transformedplants of this invention include tobacco, corn, cucumber, peppers,potatoes, soybean, squash, and tomatoes. Especially preferred aremembers of the Cucurbitaceae (e.g., squash and cucumber) family.

Another aspect of the present invention is a method of preparing aPRV-resistant plant, such as a dicot, comprising: transforming plantcells with a chimeric expression cassette comprising a promoterfunctional in plant cells operably linked to a DNA molecule that encodesa protease as described above; regenerating the plant cells to provide adifferentiated plant; and identifying a transformed plant that expressesthe PRV protease at a level sufficient to render the plant resistant toinfection by the specific strains of PRV disclosed herein.

As used herein, with respect to a DNA molecule or "gene," the phrase"isolated and purified" is defined to mean that the molecule is eitherextracted from its context in the viral genome by chemical means andpurified and/or modified to the extent that it can be introduced intothe present vectors in the appropriate orientation, i.e., sense orindecency. As used herein, the term "chimeric" refers to the linkage oftwo or more DNA molecules which are derived from different sources,strains or species (e.g., from bacteria and plants), or the linkage oftwo or more DNA molecules, which are derived from the same species andwhich are linked in a way that does not occur in the native genome. Asused herein, the term "heterologous" is defined to mean not identical,e.g. different in nucleotide and/or amino acid sequence, phenotype or anindependent isolate. As used herein, the term "expression" is defined tomean transcription or transcription followed by translation of aparticular DNA molecule. As used herein, the term "flanking genesegments" means nucleotide sequences 5' to the proteolytically derivedN-terminus of the NIa coding sequence and 3' to the proteolyticallyderived C-terminus of the NIa coding sequence, which include about 1000nucleotides each.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the genomic organization ofpotyviruses.

FIG. 2. The nucleotide sequence of the nuclear inclusion body A (NIa)protease gene and flanking gene segments of PRV FLA83 W SEQ ID NO:1!.The amino acid sequence of the encoded open reading frame is shown belowthe nucleotide sequence SEQ ID NO:2!. The nucleotide sequences ofoligonucleotides RMM354 SEQ ID NO:3! and RMM355 SEQ ID NO:4! are shownabove the nucleotide sequence, at the 5' and 3' ends of the nucleotidesequence, respectively. The viral-specific sequences in RMM354 andRMM355 are homologous to sequences in PRV HA (attenuated) USA P (Quemadaet al., J. Gen. Virol., 71, 203 (1990)). In addition RMM354 has novelrestriction endonuclease cleavage sites for EcoRI and NcoI while RMM355has novel restriction endonuclease cleavage sites for BamHI and NcoI.

FIGS. 3A-3C. The alignment of the nucleotide sequences of the nuclearinclusion body A (NIa) and flanking gene segments from PRV isolates:HA-P (Yeh et al., J. Gen. Virol., 73, 2531 (1992)) SEQ ID NO. 7!; USA P(Quemada et al., J. Gen. Virol., 71, 203 (1990) SEQ ID NO. 8!; FLA83.Sequence alignments were generated using the UWGCG program Pileup. Thedots represent either the lack of sequence information at the ends ofthe protease gene or gaps in homology in sequences relative to others inthe alignment.

FIG. 4. The alignment of the amino acid sequences from papaya ringspotvirus isolates described in FIG. 3. Sequence differences between virusstrains are underlined. The predicted cylindrical inclusion (CI)/VPg,VPg/NIa, and NIa/NIb cleavage sites are shown above the aligned aminoacids (Q/G and Q/S). Alignments were generated using the UWGCG Pileupprogram. The dots represent either the lack of sequence information atthe 5' end of the protease gene or gaps in homology in sequencesrelative to others in the alignment.

FIGS. 5A-5C. The nucleotide sequence of the nuclear inclusion body A(NIa) protease gene and flanking gene segments of PRV USA P-type strainSEQ ID NO:5!. The amino acid sequence of the encoded open reading frameis shown below the nucleotide sequence SEQ ID NO:6!. The nucleotidesequences of oligonucleotides RMM354 SEQ ID NO:3! and RMM355 SEQ IDNO:4! are shown at the 5' and 3' ends of the nucleotide sequence,respectively.

FIG. 6. A schematic representation of the cloning strategy for FLA83gene expression cassettes.

FIG. 7A and 7B. A schematic representation of the cloning strategy forPRV USA P-type (HA attenuated) gene expression cassettes.

FIGS. 8A and 8B. A schematic representation of the cloning strategy forPRV USA P-type (HA attenuated) gene expression cassettes with a stopcodon near the 5' end of the coding sequences. Oligonucleotide primersRMM333 and RMM 334 (see FIG. 5 for nucleotide sequence of these primersSEQ ID NO:9 and 10, respectively!) were used to amplify PRV USA P-type(HA attenuated) NIa sequences and introduce a stop codon into the 5'portion of the NIa gene.

DETAILED DESCRIPTION OF THE INVENTION

Papaya ringspot virus (PRV) is a single-stranded (+) RNA plant virusthat is translated into a single polyprotein. The viral RNA genome isapproximately 10,000 bases in length. The expression strategy ofpotyviruses includes translation of a complete polyprotein from thepositive sense viral genomic RNA. Translation of the genomic RNAproduces a 330 kD protein which is subsequently cleaved into at leastseven smaller viral proteins by a virally encoded protease. The virallyencoded proteins include a 35 kD protein at the amino terminal end ofthe 330 kD protein which is thought to be involved in cell to celltransmission, H C protein is 56 kD in size and is believed to beinvolved in insect transmission and possess proteolytic activity, a 50kD protein, a 90 kD cylindrical inclusion protein (CI), which is part ofthe replicase complex and possesses helicase activity, a 6 kD VPgprotein which is covalently attached to the 5' end of the viral genomicRNA, a 49 kD NIa protein which functions as a protease, a 60 kD NIbprotein which functions as a polymerase, and the coat protein (36 kD).

Two types of PRV have been established based on host range. One type isdesignated "P type"; it infects Caricacae (e.g., papaya), Cucurbitaceae(e.g., cucurbitis), and Chenopodiaceae (e.g., Chenopodium) (Wang et al.,Phytopathology, 84, 1205 (1994)). A second type is designated "W type";it infects only Cucurbitaceae and Chenopodiaceae (Wang et al.,Phytopathology, 84, 1205 (1994)). Isolates of the P type includeHA-severe, called HA-P herein (Wang et al., Phytopath., 127, 349(1992)), HA5-1, called USA P herein, YK (Wang et al., Phytopathology,84, 1205 (1994)), and other isolates as described in Tennant et al.(Phytopathology, 84, 1359 (1994)). Isolates of the W type include FLA83,disclosed herein, PRV-W type (Yeh et al., Phytopathology, 74, 1081(1984)) and PRV-W (Aust) (Bateson et al., Arch-Viol., 123, 101 (1992)).

To practice the present invention, the protease (NIa) gene of a virusmust be isolated from the viral genome and inserted into a vector. Thus,the present invention provides isolated and purified DNA molecules thatencode either an NIa protease of PRV FLA83 or an NIa protease of PRV USAP-type (HA attenuated). As used herein, a DNA molecule that encodes aprotease gene includes nucleotides of the coding strand, also referredto as the "sense" strand, as well as nucleotides of the noncodingstrand, complementary strand, also referred to as the "antisense"strand, either alone or in their base-paired configuration. For example,a DNA molecule that encodes the protease of PRV FLA83, for example,includes the DNA molecule having the nucleotide sequence of FIG. 2 SEQID NO:1!, a DNA molecule complementary to the nucleotide sequence ofFIG. 2 SEQ ID NO:1!, as well as a DNA molecule which also encodes a PRVFLA.83 NIa protease and its complement which hybridizes with a PRVFLA83-specific DNA probe in hybridization buffer with 6XSSC, 5XDenhardt's reagent, 0.5% SDS and 100 μg/ml denatured, fragmented salmonsperm DNA and remains bound when washed at 68° C. in 0.1XSSC and 0.5%SDS (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.(1989)). Moreover, the DNA molecules of the present invention caninclude non-PRV NIa protease nucleotides that do not interfere withexpression, such as the nucleotide sequences in the flanking genesegments.

The PRV protease gene does not contain the signals necessary for itsexpression once transferred and integrated into a plant genome.Accordingly, a vector must be constructed to provide the regulatorysequences such that they will be functional upon inserting a desiredgene. When the expression vector/insert construct is assembled, it isused to transform plant cells which are then used to regenerate plants.These transgenic plants carry the viral gene in the expressionvector/insert construct. The gene is expressed in the plant andincreased resistance to viral infection is conferred thereby.

Several different methods exist to isolate the protease gene. To do so,one having ordinary skill in the art can use information about thegenomic organization of potyviruses to locate and isolate the proteasegene. The protease gene is located in the 3' half of the genome, betweenthe VPg gene and the NIb gene (FIG. 1). Additionally, the informationrelated to proteolytic cleavage sites is used to determine theN-terminus of the protease gene. The protease recognition sites areconserved in the potyviruses and have been determined to be either thedipeptide Gln-Ser, Gln-Gly, or Gln-Ala. The nucleotide sequences whichencode these dipeptides can be determined.

Using methods well known in the art, a quantity of virus is grown andharvested. The viral RNA is then separated and the protease geneisolated using a number of known procedures. A cDNA library is createdusing the viral RNA, by methods known to the art. The viral RNA isincubated with primers that hybridize to the viral RNA and reversetranscriptase, and a complementary DNA molecule is produced. A DNAcomplement of the complementary DNA molecule is produced and thatsequence represents a DNA copy (cDNA) of the original viral RNAmolecule. The DNA complement can be produced in a manner that results ina single double stranded cDNA or polymerase chain reactions can be usedto amplify the DNA encoding the cDNA with the use of oligomer primersspecific for the protease gene. These primers can include novelrestriction sites used in subsequent cloning steps. Thus, a doublestranded DNA molecule is generated which contains the sequenceinformation of the viral RNA. These DNA molecules can be cloned in E.coli plasmid vectors after the additions of restriction enzyme linkermolecules by DNA ligase. The various fragments are inserted into cloningvectors, such as well-characterized plasmids, which are then used totransform E. coli and create a cDNA library.

Previously identified PRV NIa genes can be used as hybridization probesto screen the cDNA library to determine if any of the transformedbacteria contain DNA fragments with sequences coding for the proteaseregion. The cDNA inserts in any bacterial colonies which contain thisregion can be sequenced. The protease gene is present in its entirety incolonies which have sequences that extend 5' to sequences which encode aN-terminal proteolytic cleavage site and 3' to sequences which encode aC-terminal proteolytic cleavage site for NIa.

Alternatively, cDNA fragments can be inserted in the sense orientationinto expression vectors. Antibodies against the protease can be used toscreen the cDNA expression library and the gene can be isolated fromcolonies which express the protein.

Another molecular strategy to provide virus resistance in transgenicplants is based on antisense RNA. As is well known, a cell manufacturesprotein by transcribing the DNA of the gene encoding that protein toproduce RNA, which is then processed to messenger RNA (mRNA) (e.g., bythe removal of introns) and finally translated by ribosomes intoprotein. This process may be inhibited in the cell by the presense ofantisense RNA. The term antisense RNA means an RNA sequence which iscomplementary to a sequence of bases in the mRNA in question in thesense that each base (or the majority of bases) in the antisensesequence (read in the 3' to 5' sense) is capable of pairing with thecorresponding base (G with C, A with U) in the mRNA sequence read in the5' to 3' sense. It is believed that this inhibition takes place byformation of a complex between the two complementary strands of RNA,thus preventing the formation of protein. How this works is uncertain:the complex may interfere with further transcription, processing,transport or translation, or degrade the mRNA, or have more than one ofthese effects. This antisense RNA may be produced in the cell bytransformation of the cell with an appropriate DNA construct arranged totranscribe the non-template strand (as opposed to the template strand)of the relevant gene (or of a DNA sequence showing substantial homologytherewith).

The use of antisense RNA to downregulate the expression of specificplant genes is well known. Reduction of gene expression has led to achange in the phenotype of the plant: either at the level of grossvisible phenotypic difference, e.g., lack of anthocyanin production inflower petals of petunia leading to colorless instead of colored petals(van der Krol et al., Nature, 333:866-869 (1988)); or at a more subtlebiochemical level, e.g., change in the amount of polygalacturonase andreduction in depolymerization of pectin during tomato fruit ripening(Smith et al., Nature, 334:724-726 (1988)).

Another more recently described method of inhibiting gene expression intransgenic plants is the use of sense RNA transcribed from an exogenoustemplate to downregulate the expression of specific plant genes(Jorgensen, Keystone Symposium "Improved Crop and Plant Products throughBiotechnology", Abstract X1-022 (1994)). Thus, both antisense and senseRNA have been proven to be useful in achieving downregulation of geneexpression in plants.

In the present invention, the DNA molecules encoding the protease genesof PRV FLA83 W strain and the USA P-Type (HA attenuated) strain havebeen determined and the genes have been inserted into expressionvectors. These expression cassettes can be individually placed into avector that can be transmitted into plants, preferably a binary vector.Alternatively, two or more of the PRV protease genes can each be presentin an expression cassette which can be placed into the same binaryvector, or any one of the PRV NIa expression cassettes of the presentinvention can be placed into a binary vector with one or more viral geneexpression cassettes. The expression vectors contain the necessarygenetic regulatory sequences for expression of an inserted gene. Theprotease gene is inserted such that those regulatory sequences arefunctional and the genes can be expressed when incorporated into a plantgenome. For example, vectors of the present invention can containcombinations of expression cassettes that include DNA from a papayaringspot virus (PRV) coat protein gene or a heterologous PRV proteasegene (e.g., one that is from a different strain), a cucumber mosaicvirus coat protein gene, a zucchini yellow mosaic virus coat proteingene, and a watermelon mosaic virus-2 coat protein gene.

Moreover, when combinations of viral gene expression cassettes areplaced in the same binary plasmid, and that multigene cassettecontaining plasmid transformed into a plant, the multiple viral genesall preferably exhibit substantially the same degrees of efficacy whenpresent in transgenic plants. For example, if one examines numeroustransgenic lines containing two different intact viral gene expressioncassettes, the transgenic line will be immune to infection by bothviruses. Similarly, if a line exhibits a delay in symptom development toone virus, it will also exhibit a delay in symptom development to thesecond virus. Finally, if a line is susceptible to one of the viruses itwill be susceptible to the other. This phenomenon is unexpected. Ifthere were not a correlation between the performance of each gene inthese multiple gene constructs this approach as a tool in plant breedingwould probably be prohibitively difficult to use. Even with single geneconstructs, one must test numerous transgenic plant lines to find onethat displays the appropriate level of efficacy. The probability offinding a line with useful levels of expression can range from 10-50%(depending on the species involved). For further information refer toApplicants' Assignees' copending patent application Ser. No. 08/366,991entitled "Transgenic Plants Expressing DNA Constructs Containing aPlurality of Genes to Impart Virus Resistance" filed on Dec. 30, 1994,incorporated by reference herein.

In order to express the viral gene, the necessary genetic regulatorysequences must be provided. Since the protease of a potyvirus isproduced by the post-translational processing of a polyprotein, theprotease gene isolated from viral RNA does not contain transcription andtranslation signals necessary for its expression once transferred andintegrated into a plant genome. It must, therefore, be engineered tocontain a plant expressible promoter, a translation initiation codon(ATG), and a plant functional poly(A) addition signal (AATAAA) 3' of itstranslation termination codon. In the present invention, the proteasegenes are inserted into vectors which contain cloning sites forinsertion 3' of the initiation codon and 5' of the poly(A) signal. Thepromoter is 5' of the initiation codon such that when structural genesare inserted at the cloning site, a functional unit is formed in whichthe inserted genes are expressed under the control of the variousgenetic regulatory sequences.

The segment of DNA referred to as the promoter is responsible for theregulation of the transcription of DNA into mRNA. A number of promoterswhich function in plant cells are known in the art and can be employedin the practice of the present invention. These promoters can beobtained from a variety of sources such as plants or plant viruses, andcan include, but are not limited to, promoters isolated from thecaulimovirus group such as the cauliflower mosaic virus 35S promoter(CaMV35S), the enhanced cauliflower mosaic virus 35S promoter (enhCaMV35S), the figwort mosaic virus full-length transcript promoter(FMV35S), and the promoter isolated from the chlorophyll a/b bindingprotein. Other useful promoters include promoters which are capable ofexpressing the potyvirus proteins in an inducible manner or in atissue-specific manner in certain cell types in which the infection isknown to occur. For example, the inducible promoters from phenylalanineammonia lyase, chalcone synthase, hydroxyproline rich glycoprotein,extensin, pathogenesis-related proteins (e.g. PR-1a), andwound-inducible protease inhibitor from potato may be useful.

Preferred promoters for use in the present protease-containing cassettesinclude the constitutive promoters from CaMV, the Ti genes nopalinesynthase (Bevan et al., Nucleic Acids Res. II, 369 (1983)) and octopinesynthase (Depicker et al., J. Mol. Appl. Genet., 1, 561 (1982)), and thebean storage protein gene phaseolin. The poly(A) addition signals fromthese genes are also suitable for use in the present cassettes. Theparticular promoter selected is preferably capable of causing sufficientexpression of the DNA coding sequences to which it is operably linked,to result in the production of amounts of the proteins or RNAs effectiveto provide viral resistance, but not so much as to be detrimental to thecell in which they are expressed. The promoters selected should becapable of functioning in tissues including, but not limited to,epidermal, vascular, and mesophyll tissues. The actual choice of thepromoter is not critical, as long as it has sufficient transcriptionalactivity to accomplish the expression of the preselected proteins orsense and/or antisense RNAs and subsequent conferral of viral resistanceto the plants.

The nontranslated leader sequence can be derived from any suitablesource and can be specifically modified to increase the translation ofthe mRNA. The 5' nontranslated region can be obtained from the promoterselected to express the gene, an unrelated promoter, the native leadersequence of the gene or coding region to be expressed, viral RNAs,suitable eucaryotic genes, or a synthetic gene sequence. The presentinvention is not limited to the constructs presented in the followingexamples.

The termination region or 3' nontranslated region which is employed isone which will cause the termination of transcription and the additionof polyadenylated ribonucleotides to the 3' end of the transcribed mRNAsequence. The termination region can be native with the promoter region,native with the structural gene, or can be derived from another source,and preferably include a terminator and a sequence coding forpolyadenylation. Suitable 3' nontranslated regions of the chimeric plantgene include but are not limited to: (1) the 3' transcribed,nontranslated regions containing the polyadenylation signal ofAgrobacterium tumor-inducing (Ti) plasmid genes, such as the nopalinesynthase (NOS) gene; and (2) plant genes like the soybean 7S storageprotein genes.

Preferably, the expression cassettes of the present invention areengineered to contain a constitutive promoter 5' to its translationinitiation codon (ATG) and a poly(A) addition signal (AATAAA) 3' to itstranslation termination codon. Several promoters which function inplants are available, however, the preferred promoter is the 35Sconstitutive promoters from cauliflower mosaic virus (CaMV). The poly(A) signal can be obtained from the CaMV 35S gene or from any number ofwell characterized plant genes, i.e., nopaline synthase, octopinesynthase, and the bean storage protein gene phaseolin. The constructionsare similar to that used for the expression of the CMV C coat protein inPCT Patent Application PCT/US88/04321, published on Jun. 29, 1989 as WO89/05858, claiming the benefit of U.S. Ser. No. 135,591, filed Dec. 21,1987, entitled "Cucumber Mosaic Virus Coat Protein Gene", and the CMV WLcoat protein in PCT Patent Application PCT/US89/03288, published on Mar.8, 1990 as WO 90/02185, claiming the benefit of U.S. Ser. No. 234,404,filed Aug. 19, 1988, entitled "Cucumber Mosaic Virus Coat Protein Gene."

Selectable marker genes can be incorporated into the present expressioncassettes and used to select for those cells or plants which have becometransformed. The marker gene employed may express resistance to anantibiotic, such as kanamycin, gentamycin, G418, hygromycin,streptomycin, spectinomycin, tetracyline, chloramphenicol, and the like.Other markers could be employed in addition to or in the alternative,such as, for example, a gene coding for herbicide tolerance such astolerance to glyphosate, sulfonylurea, phosphinothricin, or bromoxynil.Additional means of selection could include resistance to methotrexate,heavy metals, complementation providing prototrophy to an auxotrophichost, and the like.

The particular marker employed will be one which will allow for theselection of transformed cells as opposed to those cells which are nottransformed. Depending on the number of different host species one ormore markers can be employed, where different conditions of selectionwould be useful to select the different host, and would be known tothose of skill in the art. A screenable marker such as theβ-glucuronidase gene can be used in place of, or with, a selectablemarker. Cells transformed with this gene can be identified by theproduction of a blue product on treatment with5-bromo-4-chloro-3-indoyl-β-D-glucuronide (X-Gluc).

In developing the present expression construct, i.e., expressioncassette, the various components of the expression construct such as theDNA molecules, linkers, or fragments thereof will normally be insertedinto a convenient cloning vector, such as a plasmid or phage, which iscapable of replication in a bacterial host, such as E. coli. Numerouscloning vectors exist that have been described in the literature. Aftereach cloning, the cloning vector can be isolated and subjected tofurther manipulation, such as restriction, insertion of new fragments,ligation, deletion, resection, insertion, in vitro mutagenesis, additionof polylinker fragments, and the like, in order to provide a vectorwhich will meet a particular need.

For Agrobacterium-mediated transformation, the expression cassette willbe included in a vector, and flanked by fragments of the AgrobacteriumTi or Ri plasmid, representing the right and, optionally the left,borders of the Ti or Ri plasmid transferred DNA (T-DNA). Thisfacilitates integration of the present chimeric DNA sequences into thegenome of the host plant cell. This vector will also contain sequencesthat facilitate replication of the plasmid in Agrobacterium cells, aswell as in E. coli cells.

All DNA manipulations are typically carried out in E. coli cells, andthe final plasmid bearing the potyvirus gene expression cassette ismoved into Agrobacterium cells by direct DNA transformation,conjugation, and the like. These Agrobacterium cells will contain asecond plasmid, also derived from Ti or Ri plasmids. This second plasmidwill carry all the vir genes required for transfer of the foreign DNAinto plant cells. Suitable plant transformation cloning vectors includethose derived from a Ti plasmid of Agrobacterium tumefaciens, asgenerally disclosed in Glassman et al. (U.S. Pat. No. 5,258,300), orAgrobacterium rhizogenes.

A variety of techniques are available for the introduction of thegenetic material into or transformation of the plant cell host. However,the particular manner of introduction of the plant vector into the hostis not critical to the practice of the present invention, and any methodwhich provides for efficient transformation can be employed. In additionto transformation using plant transformation vectors derived from thetumor-inducing (Ti) or root-inducing (Ri) plasmids of Agrobacterium,alternative methods could be used to insert the DNA constructs of thepresent invention into plant cells. Such methods may include, forexample, the use of liposomes, electroporation, chemicals that increasethe free uptake of DNA (Paszkowski et al., EMBO J., 3, 2717 (1984)),microinjection (Crossway et al., Mol. Gen. Genet., 202, 179 (1985)),electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA, 82, 824(1985)), or high-velocity microprojectiles (Klein et al., Nature, 327,70 (1987) and transformation using viruses or pollen.

The choice of plant tissue source or cultured plant cells fortransformation will depend on the nature of the host plant and thetransformation protocol. Useful tissue sources include callus,suspension culture cells, protoplasts, leaf segments, stem segments,tassels, pollen, embryos, hypocotyls, tuber segments, meristematicregions, and the like. The tissue source is regenerable, in that it willretain the ability to regenerate whole, fertile plants followingtransformation.

The transformation is carried out under conditions directed to the planttissue of choice. The plant cells or tissue are exposed to the DNAcarrying the present potyvirus multi-gene expression cassette for aneffective period of time. This can range from a less-than-one-secondpulse of electricity for electroporation, to a two-to-three dayco-cultivation in the presence of plasmid-bearing Agrobacterium cells.Buffers and media used will also vary with the plant tissue source andtransformation protocol. Many transformation protocols employ a feederlayer of suspended culture cells (tobacco or Black Mexican Sweet Corn,for example) on the surface of solid media plates, separated by asterile filter paper disk from the plant cells or tissues beingtransformed.

Following treatment with DNA, the plant cells or tissue may becultivated for varying lengths of time prior to selection, or may beimmediately exposed to a selective agent such as those describedhereinabove. Protocols involving exposure to Agrobacterium will alsoinclude an agent inhibitory to the growth of the Agrobacterium cells.Commonly used compounds are antibiotics such as cefotaxime andcarbenicillin. The media used in the selection may be formulated tomaintain transformed callus or suspension culture cells in anundifferentiated state, or to allow production of shoots from callus,leaf or stem segments, tuber disks, and the like.

Cells or callus observed to be growing in the presence of normallyinhibitory concentrations of the selective agents are presumed to betransformed and may be subcultured several additional times on the samemedium to remove nonresistant sections. The cells or calli can then beassayed for the presence of the viral gene cassette, or can be subjectedto known plant regeneration protocols. In protocols involving the directproduction of shoots, those shoots appearing on the selective media arepresumed to be transformed and can be excised and rooted, either onselective medium suitable for the production of roots, or by simplydipping the excised shoot in a root-inducing compound and directlyplanting it in vermiculite.

In order to produce transgenic plants exhibiting viral resistance, theviral genes must be taken up into the plant cell and stably integratedwithin the plant genome. Plant cells and tissues selected for theirresistance to an inhibitory agent are presumed to have acquired theselectable marker gene encoding this resistance during thetransformation treatment. Since the marker gene is commonly linked tothe viral genes, it can be assumed that the viral genes have similarlybeen acquired. Southern blot hybridization analysis using a probespecific to the viral genes can then be used to confirm that the foreigngenes have been taken up and integrated into the genome of the plantcell. This technique may also give some indication of the number ofcopies of the gene that have been incorporated. Successful transcriptionof the foreign gene into mRNA can likewise be assayed using Northernblot hybridization analysis of total cellular RNA and/or cellular RNAthat has been enriched in a polyadenylated region. mRNA moleculesencompassed within the scope of the invention are those which containviral specific sequences derived from the viral genes present in thetransformed vector which are of the same polarity as that of the viralgenomic RNA such that they are capable of base pairing with viralspecific RNA of the opposite polarity to that of viral genomic RNA underconditions described in Chapter 7 of Sambrook et al. (1989). Moreover,mRNA molecules encompassed within the scope of the invention are thosewhich contain viral specific sequences derived from the viral genespresent in the transformed vector which are of the opposite polarity asthat of the viral genomic RNA such that they are capable of base pairingwith viral genornic RNA under conditions described in Chapter 7 inSambrook et al. (1989).

The presence of a viral protease gene can also be detected by indirectassays, such as the Western blot assay described by Maiti et al. (Proc.Natl. Acad. Sci. U.S.A. 90, 6110 (1993)). Maiti et al. constructed afusion protein containing the TVMV NIa protease and the E. coli glnHgene and transformed the construct into tobacco. Transgenic plants wereassayed by Western blot analysis for the glnH gene product with anantibody to glnH. Not only was a glnH protein expressed in these plants,the glnH product was cleaved out of the fusion protein, presumably byNIa.

Potyvirus resistance can also be assayed via infectivity studies asgenerally disclosed by Namba et al. (Gene, 107, 181 (1991)) whereinplants are scored as symptomatic when any inoculated leaf showsveinclearing, mosaic or necrotic symptoms.

Seed from plants regenerated from tissue culture is grown in the fieldand self-pollinated to generate true breeding plants. The progeny fromthese plants become true breeding lines which are evaluated for viralresistance in the field under a range of environmental conditions. Thecommercial value of viral-resistant plants is greatest if many differenthybrid combinations with resistance are available for sale.Additionally, hybrids adapted to one part of a country are not adaptedto another part because of differences in such traits as maturity,disease and insect tolerance. Because of this, it is necessary to breedviral resistance into a large number of parental lines so that manyhybrid combinations can be produced.

Adding viral resistance to agronomically elite lines is most efficientlyaccomplished when the genetic control of viral resistance is understood.This requires crossing resistant and sensitive plants and studying thepattern of inheritance in segregating generations to ascertain whetherthe trait is expressed as dominant or recessive, the number of genesinvolved, and any possible interaction between genes if more than oneare required for expression. With respect to transgenic plants of thetype disclosed herein, the transgenes exhibit dominant, single geneMendelian behavior. This genetic analysis can be part of the initialefforts to convert agronomically elite, yet sensitive lines to resistantlines. A conversion process (backcrossing) is carried out by crossingthe original transgenic resistant line with a sensitive elite line andcrossing the progeny back to the sensitive parent. The progeny from thiscross will segregate such that some plants carry the resistance gene(s)whereas some do not. Plants carrying the resistance gene(s) will becrossed again to the sensitive parent resulting in progeny whichsegregate for resistance and sensitivity once more. This is repeateduntil the original sensitive parent has been converted to a resistantline, yet possesses all of the other important attributes originallyfound in the sensitive parent. A separate backcrossing program isimplemented for every sensitive elite line that is to be converted to avirus resistant line.

Subsequent to the backcrossing, the new resistant lines and theappropriate combinations of lines which make good commercial hybrids areevaluated for viral resistance, as well as for a battery of importantagronomic traits. Resistant lines and hybrids are produced which aretrue to type of the original sensitive lines and hybrids. This requiresevaluation under a range of environmental conditions under which thelines or hybrids will be grown commercially. Parental lines of hybridsthat perform satisfactorily are increased and utilized for hybridproduction using standard hybrid production practices.

The invention will be further described by reference to the followingdetailed examples. Enzymes were obtained from commercial sources andwere used according to the vendor's recommendations or other variationsknown in the art. Other reagents, buffers, etc., were obtained fromcommercial sources, such as GIBCO-BRL, Bethesda, Md., and Sigma ChemicalCo., St. Louis, Mo., unless otherwise specified.

Most of the recombinant DNA methods employed in practicing the presentinvention are standard procedures, well known to those skilled in theart, and described in detail in, for example, in European PatentApplication Publication Number 223,452, published Nov. 29, 1986, whichis incorporated herein by reference. General references containing suchstandard techniques include the following: R. Wu, ed., Methods inEnzymology, Vol. 68 (1979); J. H. Miller, Experiments in MolecularGenetics (1972); J. Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd ed. (1989); and D. M. Glover, ed., DNA Cloning Vol. II(1982).

FIGS. 6-8 are presented to illustrate the constructions of thisinvention.

EXAMPLE I

To isolate and engineer the PRV NIa gene, the following steps can beused. 1) Purify PRV virions and isolate PRV viral RNA from the virionpreparation; 2) construct single-stranded cDNAs of PRV viral RNA; 3)amplify NIa sequences by PCR amplification using viral sequence specificprimers; 4) clone the PCR product into a plant expression cassetteplaced into an appropriate binary vector; 5) produce PRV NIa transgenicplants; and 6) challenge the progeny of R_(o) transgenic plants toidentify lines which confer the desired properties.

A. Isolation of PRV Fla83-W Viral RNA

7-day-old yellow crookneck squash plants grown in the greenhouse wereinoculated with PRV strain W (watermelon) Florida-83; 21 days postinoculation leaves were harvested and PRV virus isolated. The procedureused was based on a modified method used by Purcifull et al.(Phytopatholoy, 69, 112 (1979)) for PRV type W isolation. Approximately50 grams of fresh leaf tissue was homogenized in 100 ml 0.5M potassiumphosphate buffer (pH 7.5 "PB") containing 0.1% sodium sulphate, 25 mlchloroform, and 25 ml carbon tetrachloride. After centrifugation of theextract at 1,000×g for 5 minutes the pellet was resuspended in 50 ml ofPB buffer and centrifuged again at 1,000×g for 5 minutes. Thesupernatants from each centrifugation are pooled then centrifuged at13,000×g for 15 minutes. To the resulting supernatant, Triton X-100 wasadded to a final concentration of 1% (v/v), polyethyleneglycol (PEG)8,000 (Reagent grade from Sigma Chemical Co.) to a final concentrationof 4% (w/v) and NaCl to a final concentration of 100 mM. The suspensionwas stirred for 1 hour at 0°-4° C. This suspension was centrifuged at10,000×g for 10 minutes.

The pellet was resuspended in 40 ml of PB. After centrifugation at12,000×g for 10 minutes the pellet was discarded and virus wasprecipitated from the supernatant by adding PEG to a final concentrationof 8% (w/v) and NaCl to a final concentration of 100 mM, and stirringfor 0.5 hour at 0°-4° C. After centrifugation at 12,000×g for 10 minutesthe pellets were resuspended with the aid of a tissue grinder in 5 ml of20 mM PB and layered over a 30% Cs₂ SO₄ cushion. This suspension wascentrifuged in a Beckman Ti75 at 140,000×g for 18 hours at 5° C. Aftercentrifugation, the virus band was harvested and dialyzed against 20 mMPB overnight at 4° C. The dialyzed virus preparation was lysed and viralRNA precipitated by the addition of LiCl to a final concentration of 2M.The viral RNA was recovered by centrifugation. Viral RNA was dissolvedand precipitated by ethanol and resuspended in water.

B. Cloning and Engineering PRV Protease Genes

(a) FLA83 W

PRV FLA83 W RNA was prepared as described above. Subsequently, the firstcDNA strand was synthesized using PRV FLA83 W RNA template in a reactionthat included the following: approximately 3-5 μg PRV FLA83 W RNA, 1 xbuffer for Superscript Reverse Transcriptase (supplied by BRL-GIBCO,Grand Island, N.Y.), 2 mM dNTPs, oligomer primer RMM355 (37.5 μg/mL, SEQID NO:4), 2.0 μL RNasin (Promega, Madison, Wis.), and 2.5 μL SuperscriptReverse Transcriptase (BRL-GIBCO) in a 20-μL reaction. After thisreaction was allowed to proceed for 30 minutes at 37° C., an aliquot ofthe first strand reaction was used as a template in a polymerase chainreaction with RMM354 and RMM 355 SEQ ID NO: 3 and 4, respectively! toamplify a region of the FLA83 W genome (FIG. 6). The RMM 354 primersupplies an ATG translation initiation codon. This region includes 189base pairs of the 3' end of the CI gene, the entire VPg gene, the entireNIa protease gene, and 146 base pairs of the 5' end of the NIb gene. The1835 bp PCR amplified product was cloned into the pCRII vector includedin the TA Cloning™ Kit supplied by Invitrogen Corp. A clone wasrecovered that contained PRV sequences (PRVNIaFLA TA-4). This clone wassequenced with the use of a kit (Sequenase 2 purchased from USB,Cleveland, Ohio).

The 1789 bp NcoI fragment of PRVNIaFLA TA-4 containing PRV sequences wasexcised from PRVNIaFLA TA-4, isolated and inserted into the plantexpression cassette pUC1318cpexpress. Cassettes containing the insert ofPRV sequences in the sense orientation were isolated by a partial BamHIdigestion (PRVFla83 NIa424) and inserted into the BglII site of pEPG111to give pEPG250 (for further information on parental binary vectorsshown in Table 1, see Applicants' Assignees' copending patentapplication Ser. No. 08/366,991 entitled "Transgenic Plants ExpressingDNA Constructs Containing a Plurality of Genes to Impart VirusResistance" filed on Dec. 30, 1994, incorporated by reference herein.For further information on PRV coat protein genes, see Applicants'Assignees' copending patent application Ser. No. 08/366,881 entitled"Papaya Ringspot Virus Coat Protein Gene" filed on Dec. 30, 1994,incorporated by reference herein. For further information on ZYMV andWMV2 coat protein genes, see Applicants' Assignees copending patentapplication Ser. No. 08/232,846 filed on Apr. 25, 1994 entitled"Potyvirus Coat Protein Genes and Plants Transformed Therewith",incorporated by reference herein. For further information of CMV-C andCMV-wl coat protein genes, see Quemada et al., J. Gen. Virol., 70, 1065(1989). The binary plasmids were transformed into Agrobacteriumtumefaciens strain C58Z707 and Mog301 (Table 1)

(b) USA Type P (HA attenuated)

A cDNA clone (#99) obtained from D. Gonsalves at Cornell University ofthe 3' end of PRV USA P-type (HA attenuated) strain served as a PCRtemplate to amplify a PRV region that included 189 bp of the 3' end ofthe CI gene, the entire VpG gene, the entire NIa protease gene, and 146bp of the 5' end of NIb gene (FIG. 5). Primers RMM354 and RMM355 SEQ IDNO: 3 and 4, respectively! were used during the PCR amplification tointroduce novel restriction sites at each end of the PRV segmentengineered. The resulting PCR-amplified segment was digested with EcoRIand BamHI (see FIGS. 2 and 5) and cloned by inserting it into the vectorpGEMX-1 (Promega, Madison, Wis.). The PRV sequences in a resulting clone(pGEMX-1 NIa-1) were nucleotide-sequenced (FIG. 5; SEQ ID NO: 5!). Therewere no sequence differences between the sequence of clone 99 and clonepGEMX-1 NIa-1.

pGEMX-1 NIa-1 was digested with NcoI and the resulting NcoI fragmentisolated for insertion into the expression cassette pUC18cpexpress. Bothsense and indecency clones of expression cassettes (the expressioncassettes are designated cpexpress PRV NIa 1-4 for the sense orientationand cpexpress PRV NIa 1-5 for the indecency orientation) containing theNcoI fragment of PRV were isolated. The plasmid containing the indecencyorientation cassette is known as pUC18cpexpressPRVNIa1-5. The plasmidcontaining the sense orientation cassette is known aspUC18cpexpressPRVNIa 1-4. Subsequently, the HindIII fragments containingexpression cassettes from each pUC18 plasmid containing eitherexpression cassette were inserted into the HindIII site of pUC1318(clone pUC1318cpexpressPRVNIa1-4 and pUC1318cpexpressPRVNIa1-5) toprovide additional sites for installing cassettes into binary plasmids(FIG. 7). Subsequently, both XbaI and BamHI fragments were isolated frompUC1318cpexpressPRVNIa1-4 and pUC1318cpexpressPRVNIa1-5. These fragmentswere inserted into the corresponding XbaI or BglII sites of pGA482G,pEPG111, pEPG106, pEPG109, pEPG120, or pEGG252 (Table 1). Resultingbinary plasmids were transformed into Agrobacteria tumefaciens strainsMog301 and C58Z707.

A PRV USA P-type (HA attenuated) NIa gene cassette was prepared thatincluded an introduced stop codon (FIG. 8). To prepare the NIa codingsequence for insertion into the expression cassette pUC18cpexpress,novel restriction sites were introduced with oligomer primers RMM333 andRMM334 (see FIG. 4 SEQ ID NO: 9 and 10, respectively!). In addition,RMM333 introduced a single base pair deletion which results in a stopcodon near the translation start site. The fragment amplified byoligomer pair RMM333 and RMM334 is 1339 base pairs in length and isshown in FIG. 8. After PCR amplification, the fragment was engineered toobtain the cassette PRVNIa-1 (stop) (HA) as shown in FIG. 8. Thecassette was inserted into binary plasmids as described in Table 1.

                  TABLE 1    ______________________________________    Binary  Parental Plasmid                        Site    PRV NIa Cassette                                          pEPG#    ______________________________________    pGA482G pEPG120     XbaI    PRVNIa1-4 200            (CMVwl62G)          (s) (HA)    pGA482G pEPG120     XbaI    PRVNIa1-5 201            (CMVwl62G)          (asdouble) (HA)    pGA482G pGA482G     XbaI    PRVNIa1-5 202                                (as) (HA)    pPRBN   pEPG109     XbaI    PRVNIa1-4 113            (CWL41/Z/W)         (s) (HA)    pPRBN   pEPG109     XbaI    PRVNIa1-5 114            (CWL41/Z/W)         (as) (HA)    pPRBN   pEPG111     BglII   PRVNIa1-4 224            (C/Z/W)             (s) (HA)    pPRBN   pEPG111     BglII   PRVNIa1-5 225            (C/Z/W)             (as) (HA)    pPRBN   pEPG106     BglII   PRVNIa1-4 226            (ZW)                (s) (HA)    pPRBN   pEPG106     BglII   PRVNIa1-5 227            (ZW)                (as) (HA)    ______________________________________

C. Transfer of PRV Protease Genes to Plants

Agrobacterium-mediated transfer of the plant expressible PRV proteasegenes described herein was done using the methods described in PCTpublished application WO 89/05859, entitled "Agrobacterium MediatedTransformation of Germinating Plant Seeds".

Transgenic cucumber lines have been produced with the USA P-type(HA-attenuated) PRV NIa gene construct described above. Progeny of R_(o)transgenic plants were challenged in the greenhouse. Challenge resultsindicate that PRV USA P-type NIa transgenic R₁ plants are protected to asignificant extent against both homologous PRV challenge andheterologous PRV challenge. Compared with non-transgenic controls,transgenic R₁ progeny show delayed onset and reduced symptoms oncucumber leaves and fruits.

All publications, patents and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 10    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1789 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (vi) ORIGINAL SOURCE:    (A) ORGANISM: PAPAYA RINGSPOT VIRUS    (B) STRAIN: W-TYPE    (C) INDIVIDUAL ISOLATE: Florida 83    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 3..1783    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 3..191    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 192..362    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 363..1643    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 1644..1783    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CCATGGGCTTCTCTCTCCTTGGTATCATAAACACTATCCAGAGTAGA47    MetGlyPheSerLeuLeuGlyIleIleAsnThrIleGlnSerArg    151015    TATTTAGTTGATCATTCAGTTGAGAATATCAGAAAGCTTCAACTAGCG95    TyrLeuValAspHisSerValGluAsnIleArgLysLeuGlnLeuAla    202530    AAGGCCCAGATTCAACAACTTGAAGCTCATGTGCAAGAGAACAATGTT143    LysAlaGlnIleGlnGlnLeuGluAlaHisValGlnGluAsnAsnVal    354045    GGAAATTTAATTCAATCTCTTGGTGCTGTCAGAGCTGTTTATCATCAA191    GlyAsnLeuIleGlnSerLeuGlyAlaValArgAlaValTyrHisGln    505560    GGTGTTGATGGAGTCAAGCACATAAAGCGAGAGTTGGGCTTGAAAGGA239    GlyValAspGlyValLysHisIleLysArgGluLeuGlyLeuLysGly    657075    GTTTGGGATGGTTCATTAATGATCAAGGATCGAATTGTATGCGGTTTC287    ValTrpAspGlySerLeuMetIleLysAspArgIleValCysGlyPhe    80859095    ACAATGGCTGGTGGTGCAATGCTCTTGTACCAACACTTTCGTGATAAG335    ThrMetAlaGlyGlyAlaMetLeuLeuTyrGlnHisPheArgAspLys    100105110    CTTACAAATGTACATGTGTTTCACCAAGGTTTCTCTGCGCGACAACGA383    LeuThrAsnValHisValPheHisGlnGlyPheSerAlaArgGlnArg    115120125    CAAAAGTTACGATTTAAGTCAGCAGCAAATGCTAAGCTTGGTCGAGAA431    GlnLysLeuArgPheLysSerAlaAlaAsnAlaLysLeuGlyArgGlu    130135140    GTCTATGGAGATGACGGGACAATTGAGCACTATTTCGGAGAAGCATAC479    ValTyrGlyAspAspGlyThrIleGluHisTyrPheGlyGluAlaTyr    145150155    ACAAAGAAAGGAAACAAGAAGGGAAAGATGCATGGCATGGGTGTTAAA527    ThrLysLysGlyAsnLysLysGlyLysMetHisGlyMetGlyValLys    160165170175    ACGAGAAAGTTCGTTGCAACATATGGATTTAAACCAGAGGATTATTCA575    ThrArgLysPheValAlaThrTyrGlyPheLysProGluAspTyrSer    180185190    TACGTGCGGTACTTGGATCCTTTAACAGGTGAGACTTTGGATGAAAGC623    TyrValArgTyrLeuAspProLeuThrGlyGluThrLeuAspGluSer    195200205    CCACAGACTGACATCTCAATGGTGCAAGAACATTTTGGTGATATTCGG671    ProGlnThrAspIleSerMetValGlnGluHisPheGlyAspIleArg    210215220    AGTAAATATTTGGATTCAGACAGCTTCGACAGGCAGGCTTTAATAGCA719    SerLysTyrLeuAspSerAspSerPheAspArgGlnAlaLeuIleAla    225230235    AACAATACAATTAAGGCCTATTATGTCCGAAACTCCGCGAAGACAGCA767    AsnAsnThrIleLysAlaTyrTyrValArgAsnSerAlaLysThrAla    240245250255    TTGGAAGTCGATTTGACACCGCATAACCCTCTGAAAGTTTGTGACAAC815    LeuGluValAspLeuThrProHisAsnProLeuLysValCysAspAsn    260265270    AAATTGACTATTGCAGGATTTCCTGATAGAGAAGCTGAACTGAGACAA863    LysLeuThrIleAlaGlyPheProAspArgGluAlaGluLeuArgGln    275280285    ACAGGCCCAGCCAGAACTATTCAAGCCGATCAAGTTCCACCACCTTCG911    ThrGlyProAlaArgThrIleGlnAlaAspGlnValProProProSer    290295300    AAATCAGTTCATCACGAAGGAAAAAGTCTTTGTCAAGGTATGAGAAAT959    LysSerValHisHisGluGlyLysSerLeuCysGlnGlyMetArgAsn    305310315    TACAATGGCATAGCTTCCGTGGTTTGCCATTTGAAAAACACATCGGGA1007    TyrAsnGlyIleAlaSerValValCysHisLeuLysAsnThrSerGly    320325330335    GATGGGAGAAGCCTATTTGGAATCGGATATAACTCGTTCATCATTACA1055    AspGlyArgSerLeuPheGlyIleGlyTyrAsnSerPheIleIleThr    340345350    AACCGACATTTGTTCAAAGAAAATAATGGTGAACTTATAGTGAAATCC1103    AsnArgHisLeuPheLysGluAsnAsnGlyGluLeuIleValLysSer    355360365    CAACACGGCAAGTTTGTTGTCAAGAACACCTCAACGCTCCGAATTGCT1151    GlnHisGlyLysPheValValLysAsnThrSerThrLeuArgIleAla    370375380    CCAGTTGGAAAAACTGATCTTTTGATAATTCGGATGCCGAAAGACTTT1199    ProValGlyLysThrAspLeuLeuIleIleArgMetProLysAspPhe    385390395    CCTCCATTCCATAGTAGAGCTAGGTTTAGGGCCATGAAAGCTGGAGAC1247    ProProPheHisSerArgAlaArgPheArgAlaMetLysAlaGlyAsp    400405410415    AAGGTTTGCATGATCGGTGTTGACTACCAAGAGAATCATATTGCGAGC1295    LysValCysMetIleGlyValAspTyrGlnGluAsnHisIleAlaSer    420425430    AAAGTATCTGAAACTTCTATTATCAGTGAGGGCACGGGAGAGTTTGGA1343    LysValSerGluThrSerIleIleSerGluGlyThrGlyGluPheGly    435440445    TGCCATTGGATATCCACGAATGATGGTGATTGCGGTAATCCACTAGTT1391    CysHisTrpIleSerThrAsnAspGlyAspCysGlyAsnProLeuVal    450455460    AGTGTTTCAGATGGTTTCATTGTTGGCTTGCATAGTTTGTCGACATCA1439    SerValSerAspGlyPheIleValGlyLeuHisSerLeuSerThrSer    465470475    ACCGGAAATCAAAATTTCTTCGCTAAAATACCCGCACAATTTGAAGAA1487    ThrGlyAsnGlnAsnPhePheAlaLysIleProAlaGlnPheGluGlu    480485490495    AAGGTCCTGAGGAAAATTGATGAATTAACATGGAGCAAACACTGGAGC1535    LysValLeuArgLysIleAspGluLeuThrTrpSerLysHisTrpSer    500505510    TACAATATTAATGAACTGAGTTGGGGAGCTCTTAAGGTGTGGGAAAGT1583    TyrAsnIleAsnGluLeuSerTrpGlyAlaLeuLysValTrpGluSer    515520525    CGTCCCGAAGCAATTTTTAATGCGCAAAAGGAAGTCAACCAATTGAAT1631    ArgProGluAlaIlePheAsnAlaGlnLysGluValAsnGlnLeuAsn    530535540    GTTTTTGAGCAAAGTGGTAGTCGTTGGCTCTTCGACAAATTACACGGC1679    ValPheGluGlnSerGlySerArgTrpLeuPheAspLysLeuHisGly    545550555    AATTTGAAGGGTGTAAGTTCCGCTTCTAGCAATTTGGTGACAAAGCAC1727    AsnLeuLysGlyValSerSerAlaSerSerAsnLeuValThrLysHis    560565570575    GTTGTTAAAGGCATTTGTCCTCTCTTCAGGAACTATCTCGAGTGTGAT1775    ValValLysGlyIleCysProLeuPheArgAsnTyrLeuGluCysAsp    580585590    GAATAGGCCCATGG1789    Glu*    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 592 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetGlyPheSerLeuLeuGlyIleIleAsnThrIleGlnSerArgTyr    151015    LeuValAspHisSerValGluAsnIleArgLysLeuGlnLeuAlaLys    202530    AlaGlnIleGlnGlnLeuGluAlaHisValGlnGluAsnAsnValGly    354045    AsnLeuIleGlnSerLeuGlyAlaValArgAlaValTyrHisGlnGly    505560    ValAspGlyValLysHisIleLysArgGluLeuGlyLeuLysGlyVal    65707580    TrpAspGlySerLeuMetIleLysAspArgIleValCysGlyPheThr    859095    MetAlaGlyGlyAlaMetLeuLeuTyrGlnHisPheArgAspLysLeu    100105110    ThrAsnValHisValPheHisGlnGlyPheSerAlaArgGlnArgGln    115120125    LysLeuArgPheLysSerAlaAlaAsnAlaLysLeuGlyArgGluVal    130135140    TyrGlyAspAspGlyThrIleGluHisTyrPheGlyGluAlaTyrThr    145150155160    LysLysGlyAsnLysLysGlyLysMetHisGlyMetGlyValLysThr    165170175    ArgLysPheValAlaThrTyrGlyPheLysProGluAspTyrSerTyr    180185190    ValArgTyrLeuAspProLeuThrGlyGluThrLeuAspGluSerPro    195200205    GlnThrAspIleSerMetValGlnGluHisPheGlyAspIleArgSer    210215220    LysTyrLeuAspSerAspSerPheAspArgGlnAlaLeuIleAlaAsn    225230235240    AsnThrIleLysAlaTyrTyrValArgAsnSerAlaLysThrAlaLeu    245250255    GluValAspLeuThrProHisAsnProLeuLysValCysAspAsnLys    260265270    LeuThrIleAlaGlyPheProAspArgGluAlaGluLeuArgGlnThr    275280285    GlyProAlaArgThrIleGlnAlaAspGlnValProProProSerLys    290295300    SerValHisHisGluGlyLysSerLeuCysGlnGlyMetArgAsnTyr    305310315320    AsnGlyIleAlaSerValValCysHisLeuLysAsnThrSerGlyAsp    325330335    GlyArgSerLeuPheGlyIleGlyTyrAsnSerPheIleIleThrAsn    340345350    ArgHisLeuPheLysGluAsnAsnGlyGluLeuIleValLysSerGln    355360365    HisGlyLysPheValValLysAsnThrSerThrLeuArgIleAlaPro    370375380    ValGlyLysThrAspLeuLeuIleIleArgMetProLysAspPhePro    385390395400    ProPheHisSerArgAlaArgPheArgAlaMetLysAlaGlyAspLys    405410415    ValCysMetIleGlyValAspTyrGlnGluAsnHisIleAlaSerLys    420425430    ValSerGluThrSerIleIleSerGluGlyThrGlyGluPheGlyCys    435440445    HisTrpIleSerThrAsnAspGlyAspCysGlyAsnProLeuValSer    450455460    ValSerAspGlyPheIleValGlyLeuHisSerLeuSerThrSerThr    465470475480    GlyAsnGlnAsnPhePheAlaLysIleProAlaGlnPheGluGluLys    485490495    ValLeuArgLysIleAspGluLeuThrTrpSerLysHisTrpSerTyr    500505510    AsnIleAsnGluLeuSerTrpGlyAlaLeuLysValTrpGluSerArg    515520525    ProGluAlaIlePheAsnAlaGlnLysGluValAsnGlnLeuAsnVal    530535540    PheGluGlnSerGlySerArgTrpLeuPheAspLysLeuHisGlyAsn    545550555560    LeuLysGlyValSerSerAlaSerSerAsnLeuValThrLysHisVal    565570575    ValLysGlyIleCysProLeuPheArgAsnTyrLeuGluCysAspGlu    580585590    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 43 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "synthetic oligonucleotide"    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GCTATGACAGAATTCACTGGCCTAACCATGGGCTTCTCTCTCC43    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 42 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "synthetic oligonucleotide"    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    CCCATAAGTGGATCCAAGAAACCATGGGCCTATTCATCACAC42    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1797 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (vi) ORIGINAL SOURCE:    (A) ORGANISM: PAPAYA RINGSPOT VIRUS    (B) STRAIN: P-TYPE    (C) INDIVIDUAL ISOLATE: USA (HA attenuated)    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 3..1782    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 3..191    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 192..362    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 363..1643    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 1644..1782    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CCATGGGCTTCTCTCTCCTTGGTGTTATAAACACTATCCAGAGTAGA47    MetGlyPheSerLeuLeuGlyValIleAsnThrIleGlnSerArg    151015    TATCTAGTTGACCACTCAGTTGAAAATATCAGAAAACTTCAACTGGCG95    TyrLeuValAspHisSerValGluAsnIleArgLysLeuGlnLeuAla    202530    AAGGCCCAAATTCAACAACTTGAAGCTCATGTGCAGGAAAACAATGTT143    LysAlaGlnIleGlnGlnLeuGluAlaHisValGlnGluAsnAsnVal    354045    GAAAATTTAATTCAATCTCTTGGTGCTGTCAGAGCTGTTTACCATCAA191    GluAsnLeuIleGlnSerLeuGlyAlaValArgAlaValTyrHisGln    505560    AGTGTTGATGGATTTAAACACATAAAGCGAGAGTTGGGTTTGAAAGGA239    SerValAspGlyPheLysHisIleLysArgGluLeuGlyLeuLysGly    657075    GTTTGGGATGGCTCATTGATGATTAAGGATGCGATTGTATGCGGTTTC287    ValTrpAspGlySerLeuMetIleLysAspAlaIleValCysGlyPhe    80859095    ACAATGGCTGGCGGTGCGATGCTTTTGTACCAACATTTTCGTGATAAG335    ThrMetAlaGlyGlyAlaMetLeuLeuTyrGlnHisPheArgAspLys    100105110    TTTACAAATGTTCATGTGTTTCACCAAGGTTTCTCTGCGCGACAGAGA383    PheThrAsnValHisValPheHisGlnGlyPheSerAlaArgGlnArg    115120125    CAAAAGTTAAGATTTAAGTCAGCAGCGAATGCTAAGCTTGGTCGAGAG431    GlnLysLeuArgPheLysSerAlaAlaAsnAlaLysLeuGlyArgGlu    130135140    GTCTATGGAGATGATGGGACAATTGAGCACTATTTTGGAGAAGCGTAC479    ValTyrGlyAspAspGlyThrIleGluHisTyrPheGlyGluAlaTyr    145150155    ACGAAGAAAGGAAACAAGAAAGGAAAGATGCATGGCATGGGTGTTAAG527    ThrLysLysGlyAsnLysLysGlyLysMetHisGlyMetGlyValLys    160165170175    ACGAGAAAGTTTGTTGCGACATATGGATTTAAACCGGAGGATTACTCG575    ThrArgLysPheValAlaThrTyrGlyPheLysProGluAspTyrSer    180185190    TACGTGCGGTACTTGGACCCTTTAACAGGTGAGACTTTGGATGAAAGC623    TyrValArgTyrLeuAspProLeuThrGlyGluThrLeuAspGluSer    195200205    CCACAGACTGATATCTCAATGGTGCAAGATCATTTTAGTGATATTCGG671    ProGlnThrAspIleSerMetValGlnAspHisPheSerAspIleArg    210215220    AGAAAGTACATGGATTCAGACAGCTTCGATAGGCAGGCTTTAATAGCA719    ArgLysTyrMetAspSerAspSerPheAspArgGlnAlaLeuIleAla    225230235    AACAATACAATTAAGGCTTATTATGTCCGAAACTCCGCGAAGGCAGCA767    AsnAsnThrIleLysAlaTyrTyrValArgAsnSerAlaLysAlaAla    240245250255    TTGGAAGTCGATCTGACACCGCACAACCCTCTCAAAGTTTGTGACAAT815    LeuGluValAspLeuThrProHisAsnProLeuLysValCysAspAsn    260265270    AAATTGACCATTGCAGGATTTCCTGACAGGGAAGCTGAGCTGAGACAA863    LysLeuThrIleAlaGlyPheProAspArgGluAlaGluLeuArgGln    275280285    ACAGGCCCGCCCAGAACTATTCAAGTAGATCAAGTGCCACCACCCTCG911    ThrGlyProProArgThrIleGlnValAspGlnValProProProSer    290295300    AAATCAGTTCATCACGAAGGAAAAAGTCTTTGTCAAGGCATGAGAAAT959    LysSerValHisHisGluGlyLysSerLeuCysGlnGlyMetArgAsn    305310315    TACAATGGCATAGCTTCTGTGGTTTGCCATTTGAAAAACACATCAGGA1007    TyrAsnGlyIleAlaSerValValCysHisLeuLysAsnThrSerGly    320325330335    AAGGGAAAGAGCTTGTTTGGAATTGGATATAATTCATTCATCATTACC1055    LysGlyLysSerLeuPheGlyIleGlyTyrAsnSerPheIleIleThr    340345350    AACCGACATTTGTTCAAGGAGAATAATGGTGAACTTATAGTGAAATCC1103    AsnArgHisLeuPheLysGluAsnAsnGlyGluLeuIleValLysSer    355360365    CAACACGGTAAGTTTATTGTCAAGAACACCACAACACTCCGAATTGCT1151    GlnHisGlyLysPheIleValLysAsnThrThrThrLeuArgIleAla    370375380    CCAGTTGGAAAGACTGATCTTTTAATTATTCGGATGCCGAAAGATTTT1199    ProValGlyLysThrAspLeuLeuIleIleArgMetProLysAspPhe    385390395    CCTCCATTCCATAGCAGAGCTAGGTTTAGGGCCATGAAAGCTGGGGAC1247    ProProPheHisSerArgAlaArgPheArgAlaMetLysAlaGlyAsp    400405410415    AAGGTTTGCATGATAGGTGTTGACTACCAAGAGAATCATATCGCGAGC1295    LysValCysMetIleGlyValAspTyrGlnGluAsnHisIleAlaSer    420425430    AAAGTATCTGAAACCTCTATCATCAGTGAGGGCACGGGAGATTTTGGA1343    LysValSerGluThrSerIleIleSerGluGlyThrGlyAspPheGly    435440445    TGCCACTGGATATCCACGAATGACGGTGATTGCGGTAATCCTTTAGTT1391    CysHisTrpIleSerThrAsnAspGlyAspCysGlyAsnProLeuVal    450455460    AGTGTTTCAGATGGTTTTATTGTCGGCTTGCATAGTTTGTCGACATCA1439    SerValSerAspGlyPheIleValGlyLeuHisSerLeuSerThrSer    465470475    ACTGGAGATCAAAATTTCTTTGCTAAAATACCCGCACAATTTGAAGAA1487    ThrGlyAspGlnAsnPhePheAlaLysIleProAlaGlnPheGluGlu    480485490495    AAGGTCCTTAGGAAGATTGATGATTTAACTTGGAGCAAACACTGGAGC1535    LysValLeuArgLysIleAspAspLeuThrTrpSerLysHisTrpSer    500505510    TATAATATTAATGAACTGAGTTGGGGAGCTCTCAAAGTGTGGGAAAGT1583    TyrAsnIleAsnGluLeuSerTrpGlyAlaLeuLysValTrpGluSer    515520525    CGGCCCGAAGCAATTTTTAACGCGCAAAAGGAAGTTAATCAATTGAAT1631    ArgProGluAlaIlePheAsnAlaGlnLysGluValAsnGlnLeuAsn    530535540    GTTTTCGAGCAAAGTGGTAGTCGTTGGCTCTTTGACAAATTACACGGC1679    ValPheGluGlnSerGlySerArgTrpLeuPheAspLysLeuHisGly    545550555    AATTTGAAAGGAGTTAGCTCCGCTCCTAGCAATTTGGTGACAAAGCAC1727    AsnLeuLysGlyValSerSerAlaProSerAsnLeuValThrLysHis    560565570575    GTTGTTAAAGGAATTTGTCCTCTTTTCAGGAACTATCTCGAGTGTGAT1775    ValValLysGlyIleCysProLeuPheArgAsnTyrLeuGluCysAsp    580585590    GAATAGGCCCATGGTTGCGCTG1797    Glu*    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 592 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    MetGlyPheSerLeuLeuGlyValIleAsnThrIleGlnSerArgTyr    151015    LeuValAspHisSerValGluAsnIleArgLysLeuGlnLeuAlaLys    202530    AlaGlnIleGlnGlnLeuGluAlaHisValGlnGluAsnAsnValGlu    354045    AsnLeuIleGlnSerLeuGlyAlaValArgAlaValTyrHisGlnSer    505560    ValAspGlyPheLysHisIleLysArgGluLeuGlyLeuLysGlyVal    65707580    TrpAspGlySerLeuMetIleLysAspAlaIleValCysGlyPheThr    859095    MetAlaGlyGlyAlaMetLeuLeuTyrGlnHisPheArgAspLysPhe    100105110    ThrAsnValHisValPheHisGlnGlyPheSerAlaArgGlnArgGln    115120125    LysLeuArgPheLysSerAlaAlaAsnAlaLysLeuGlyArgGluVal    130135140    TyrGlyAspAspGlyThrIleGluHisTyrPheGlyGluAlaTyrThr    145150155160    LysLysGlyAsnLysLysGlyLysMetHisGlyMetGlyValLysThr    165170175    ArgLysPheValAlaThrTyrGlyPheLysProGluAspTyrSerTyr    180185190    ValArgTyrLeuAspProLeuThrGlyGluThrLeuAspGluSerPro    195200205    GlnThrAspIleSerMetValGlnAspHisPheSerAspIleArgArg    210215220    LysTyrMetAspSerAspSerPheAspArgGlnAlaLeuIleAlaAsn    225230235240    AsnThrIleLysAlaTyrTyrValArgAsnSerAlaLysAlaAlaLeu    245250255    GluValAspLeuThrProHisAsnProLeuLysValCysAspAsnLys    260265270    LeuThrIleAlaGlyPheProAspArgGluAlaGluLeuArgGlnThr    275280285    GlyProProArgThrIleGlnValAspGlnValProProProSerLys    290295300    SerValHisHisGluGlyLysSerLeuCysGlnGlyMetArgAsnTyr    305310315320    AsnGlyIleAlaSerValValCysHisLeuLysAsnThrSerGlyLys    325330335    GlyLysSerLeuPheGlyIleGlyTyrAsnSerPheIleIleThrAsn    340345350    ArgHisLeuPheLysGluAsnAsnGlyGluLeuIleValLysSerGln    355360365    HisGlyLysPheIleValLysAsnThrThrThrLeuArgIleAlaPro    370375380    ValGlyLysThrAspLeuLeuIleIleArgMetProLysAspPhePro    385390395400    ProPheHisSerArgAlaArgPheArgAlaMetLysAlaGlyAspLys    405410415    ValCysMetIleGlyValAspTyrGlnGluAsnHisIleAlaSerLys    420425430    ValSerGluThrSerIleIleSerGluGlyThrGlyAspPheGlyCys    435440445    HisTrpIleSerThrAsnAspGlyAspCysGlyAsnProLeuValSer    450455460    ValSerAspGlyPheIleValGlyLeuHisSerLeuSerThrSerThr    465470475480    GlyAspGlnAsnPhePheAlaLysIleProAlaGlnPheGluGluLys    485490495    ValLeuArgLysIleAspAspLeuThrTrpSerLysHisTrpSerTyr    500505510    AsnIleAsnGluLeuSerTrpGlyAlaLeuLysValTrpGluSerArg    515520525    ProGluAlaIlePheAsnAlaGlnLysGluValAsnGlnLeuAsnVal    530535540    PheGluGlnSerGlySerArgTrpLeuPheAspLysLeuHisGlyAsn    545550555560    LeuLysGlyValSerSerAlaProSerAsnLeuValThrLysHisVal    565570575    ValLysGlyIleCysProLeuPheArgAsnTyrLeuGluCysAspGlu    580585590    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1900 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (vi) ORIGINAL SOURCE:    (A) ORGANISM: PAPAYA RINGSPOT VIRUS    (B) STRAIN: P-TYPE    (C) INDIVIDUAL ISOLATE: Hawaii    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 3..1900    (x) PUBLICATION INFORMATION:    (A) AUTHORS: Yeh, SD    Jan, F    Chiang, C    Doong, T    Chen, M    Chung, P    Bau, H    (B) TITLE: Complete nucleotide sequence and genetic    organization of papaya ringspot virus.    (C) JOURNAL: J. Gen. Virol.    (D) VOLUME: 73    (F) PAGES: 2531-    (G) DATE: 1992    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GCACTGGCCTAAACTCTAGCTTCTCTCTCCTTGGTGTTATAAACACT47    ThrGlyLeuAsnSerSerPheSerLeuLeuGlyValIleAsnThr    595600605    ATCCAGAGTAGATATCTAGTTGACCACTCAGTTGAAAATATCAGAAAA95    IleGlnSerArgTyrLeuValAspHisSerValGluAsnIleArgLys    610615620    CTTCAACTGGCAAAGGCCCAGATTCAACAACTTGAAGCTCACATGCAG143    LeuGlnLeuAlaLysAlaGlnIleGlnGlnLeuGluAlaHisMetGln    625630635640    GAAAACAATGTTGAAAATTTAATTCAATCTCTTGGTGCTGTAAGAGCT191    GluAsnAsnValGluAsnLeuIleGlnSerLeuGlyAlaValArgAla    645650655    GTTTACCATCAAAGTGTTGATGGATTTAAACACATAAAGCGAGAGTTG239    ValTyrHisGlnSerValAspGlyPheLysHisIleLysArgGluLeu    660665670    GGTTTGAAAGGAGTTTGGGATGGCTCATTGATGATTAAGGATGCGATT287    GlyLeuLysGlyValTrpAspGlySerLeuMetIleLysAspAlaIle    675680685    GTATGCGGTTTCACAATGGCTGGCGGTGCGATGCTTTTGTACCAACAC335    ValCysGlyPheThrMetAlaGlyGlyAlaMetLeuLeuTyrGlnHis    690695700    TTTCGTGATAAGTTTACAAATGTTCATGTGTTTCACCAAGGTTTCTCT383    PheArgAspLysPheThrAsnValHisValPheHisGlnGlyPheSer    705710715720    GCGCGACAGAGACAAAAGTTAAGATTTAAGTCAGCAGCGAATGCTAAG431    AlaArgGlnArgGlnLysLeuArgPheLysSerAlaAlaAsnAlaLys    725730735    CTTGGTCGAGAGGTCTATGGAGATGATGGGACAATTGAGCACTATTTT479    LeuGlyArgGluValTyrGlyAspAspGlyThrIleGluHisTyrPhe    740745750    GGAGAAGCGTACACGAAGAAAGGAAACAAGAAAGGAAAGATGCATGGC527    GlyGluAlaTyrThrLysLysGlyAsnLysLysGlyLysMetHisGly    755760765    ATGGGTGTTAAGACGAGAAAGTTTGTTGCGACATATGGATTTAAACCG575    MetGlyValLysThrArgLysPheValAlaThrTyrGlyPheLysPro    770775780    GAGGATTACTCGTACGTGCGGTACTTGGACCCTTTAACAGGTGAGACT623    GluAspTyrSerTyrValArgTyrLeuAspProLeuThrGlyGluThr    785790795800    TTGGATGAAAGCCCACAGACTGATATCTCAATGGTGCAAGATCATTTT671    LeuAspGluSerProGlnThrAspIleSerMetValGlnAspHisPhe    805810815    AGTGATATTCGGAGAAAGTACATGGATTCAGACAGCTTCGATAGGCAG719    SerAspIleArgArgLysTyrMetAspSerAspSerPheAspArgGln    820825830    GCTTTAATAGCAAACAATACAATTAAGGCTTATTATGTCCGAAACTCC767    AlaLeuIleAlaAsnAsnThrIleLysAlaTyrTyrValArgAsnSer    835840845    GCGAAGGCAGCATTGGAAGTCGATCTGACACCGCACAACCCTCTCAAA815    AlaLysAlaAlaLeuGluValAspLeuThrProHisAsnProLeuLys    850855860    GTTTGTGACAATAAATTGACCATTGCAGGATTTCCTGACAGGGAAGCT863    ValCysAspAsnLysLeuThrIleAlaGlyPheProAspArgGluAla    865870875880    GAGCTAAGACAAACAGGCCCGCCCAGAACTATTCAAGTAGATCAAGTG911    GluLeuArgGlnThrGlyProProArgThrIleGlnValAspGlnVal    885890895    CCACCACCCTCGAAATCAGTTCATCACGAAGGAAAAAGTCTTTGTCAA959    ProProProSerLysSerValHisHisGluGlyLysSerLeuCysGln    900905910    GGCATGAGAAATTACAATGGCATAGCTTCTGTGGTTTGCCATTTGAAA1007    GlyMetArgAsnTyrAsnGlyIleAlaSerValValCysHisLeuLys    915920925    AACACATCAGGAAAGGGGAAGAGCTTGTTTGGAATTGGATATAATTCA1055    AsnThrSerGlyLysGlyLysSerLeuPheGlyIleGlyTyrAsnSer    930935940    TTCATCATTACCAACCGACATTTGTTCAAGGAGAATAATGGTGAACTT1103    PheIleIleThrAsnArgHisLeuPheLysGluAsnAsnGlyGluLeu    945950955960    ATAGTGAAATCCCAACACGGTAAGTTTATTGTCAAGAACACCACAACA1151    IleValLysSerGlnHisGlyLysPheIleValLysAsnThrThrThr    965970975    CTCCAAATTGCTCCAGTTGGAAAGACTGATCTTTTAATTATTCGGATG1199    LeuGlnIleAlaProValGlyLysThrAspLeuLeuIleIleArgMet    980985990    CCGAAAGATTTTCCTCCATTCCATAGCAGAGCTAGGTTTAGGGCCATG1247    ProLysAspPheProProPheHisSerArgAlaArgPheArgAlaMet    99510001005    AAAGCTGGGGACAAGGTTTGCATGATAGGTGTTGACTACCAAGAGAAT1295    LysAlaGlyAspLysValCysMetIleGlyValAspTyrGlnGluAsn    101010151020    CATATCGCGAGCAAAGTATCTGAAACCTCTATCATCAGTGAGGGCACG1343    HisIleAlaSerLysValSerGluThrSerIleIleSerGluGlyThr    1025103010351040    GGAGATTTTGGATGCCACTGGATATCCACGAATGACGGTGATTGCGGT1391    GlyAspPheGlyCysHisTrpIleSerThrAsnAspGlyAspCysGly    104510501055    AATCCTTTAGTTAGTGTTTCAGATGGTTTTATTGTCGGCTTGCATAGT1439    AsnProLeuValSerValSerAspGlyPheIleValGlyLeuHisSer    106010651070    TTGTCGACATCAACTGGAGATCAAAATTTCTTTGCCAAAATACCCGCA1487    LeuSerThrSerThrGlyAspGlnAsnPhePheAlaLysIleProAla    107510801085    CAATTTGAAGAAAAGGTCCTTAGGAAGATTGATGATTTAACTTGGAGC1535    GlnPheGluGluLysValLeuArgLysIleAspAspLeuThrTrpSer    109010951100    AAACACTGGAGCTATAATATTAATGAACTGAGTTGGGGAGCTCTCAAA1583    LysHisTrpSerTyrAsnIleAsnGluLeuSerTrpGlyAlaLeuLys    1105111011151120    GTGTGGGAAAGTCGGCCCGAAGCAATTTTTAACGCACAAAAGGAAGTT1631    ValTrpGluSerArgProGluAlaIlePheAsnAlaGlnLysGluVal    112511301135    AATCAATTGAATGTTTTCGAGCAAAGTGGTGGTCGTTGGCTCTTTGAC1679    AsnGlnLeuAsnValPheGluGlnSerGlyGlyArgTrpLeuPheAsp    114011451150    AAATTACACGGCAATTTGAAAGGAGTTAGCTCCGCTCCTAGCAATTTG1727    LysLeuHisGlyAsnLeuLysGlyValSerSerAlaProSerAsnLeu    115511601165    GTGACAAAGCACGTTGTTAAAGGAATTTGTCCTCTTTTCAGGAACTAT1775    ValThrLysHisValValLysGlyIleCysProLeuPheArgAsnTyr    117011751180    CTCGAGTGTGATGAAGAGGCTAAAGCTTTCTTTAGTCCACTTATGGGT1823    LeuGluCysAspGluGluAlaLysAlaPhePheSerProLeuMetGly    1185119011951200    CACTACATGAAGAGTGTTCTGAGCAAGGAAGCGTACATTAAGGATTTA1871    HisTyrMetLysSerValLeuSerLysGluAlaTyrIleLysAspLeu    120512101215    TTGAAATATTCAAGTGATATTGTCGTTGG1900    LeuLysTyrSerSerAspIleValVal    12201225    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 632 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    ThrGlyLeuAsnSerSerPheSerLeuLeuGlyValIleAsnThrIle    151015    GlnSerArgTyrLeuValAspHisSerValGluAsnIleArgLysLeu    202530    GlnLeuAlaLysAlaGlnIleGlnGlnLeuGluAlaHisMetGlnGlu    354045    AsnAsnValGluAsnLeuIleGlnSerLeuGlyAlaValArgAlaVal    505560    TyrHisGlnSerValAspGlyPheLysHisIleLysArgGluLeuGly    65707580    LeuLysGlyValTrpAspGlySerLeuMetIleLysAspAlaIleVal    859095    CysGlyPheThrMetAlaGlyGlyAlaMetLeuLeuTyrGlnHisPhe    100105110    ArgAspLysPheThrAsnValHisValPheHisGlnGlyPheSerAla    115120125    ArgGlnArgGlnLysLeuArgPheLysSerAlaAlaAsnAlaLysLeu    130135140    GlyArgGluValTyrGlyAspAspGlyThrIleGluHisTyrPheGly    145150155160    GluAlaTyrThrLysLysGlyAsnLysLysGlyLysMetHisGlyMet    165170175    GlyValLysThrArgLysPheValAlaThrTyrGlyPheLysProGlu    180185190    AspTyrSerTyrValArgTyrLeuAspProLeuThrGlyGluThrLeu    195200205    AspGluSerProGlnThrAspIleSerMetValGlnAspHisPheSer    210215220    AspIleArgArgLysTyrMetAspSerAspSerPheAspArgGlnAla    225230235240    LeuIleAlaAsnAsnThrIleLysAlaTyrTyrValArgAsnSerAla    245250255    LysAlaAlaLeuGluValAspLeuThrProHisAsnProLeuLysVal    260265270    CysAspAsnLysLeuThrIleAlaGlyPheProAspArgGluAlaGlu    275280285    LeuArgGlnThrGlyProProArgThrIleGlnValAspGlnValPro    290295300    ProProSerLysSerValHisHisGluGlyLysSerLeuCysGlnGly    305310315320    MetArgAsnTyrAsnGlyIleAlaSerValValCysHisLeuLysAsn    325330335    ThrSerGlyLysGlyLysSerLeuPheGlyIleGlyTyrAsnSerPhe    340345350    IleIleThrAsnArgHisLeuPheLysGluAsnAsnGlyGluLeuIle    355360365    ValLysSerGlnHisGlyLysPheIleValLysAsnThrThrThrLeu    370375380    GlnIleAlaProValGlyLysThrAspLeuLeuIleIleArgMetPro    385390395400    LysAspPheProProPheHisSerArgAlaArgPheArgAlaMetLys    405410415    AlaGlyAspLysValCysMetIleGlyValAspTyrGlnGluAsnHis    420425430    IleAlaSerLysValSerGluThrSerIleIleSerGluGlyThrGly    435440445    AspPheGlyCysHisTrpIleSerThrAsnAspGlyAspCysGlyAsn    450455460    ProLeuValSerValSerAspGlyPheIleValGlyLeuHisSerLeu    465470475480    SerThrSerThrGlyAspGlnAsnPhePheAlaLysIleProAlaGln    485490495    PheGluGluLysValLeuArgLysIleAspAspLeuThrTrpSerLys    500505510    HisTrpSerTyrAsnIleAsnGluLeuSerTrpGlyAlaLeuLysVal    515520525    TrpGluSerArgProGluAlaIlePheAsnAlaGlnLysGluValAsn    530535540    GlnLeuAsnValPheGluGlnSerGlyGlyArgTrpLeuPheAspLys    545550555560    LeuHisGlyAsnLeuLysGlyValSerSerAlaProSerAsnLeuVal    565570575    ThrLysHisValValLysGlyIleCysProLeuPheArgAsnTyrLeu    580585590    GluCysAspGluGluAlaLysAlaPhePheSerProLeuMetGlyHis    595600605    TyrMetLysSerValLeuSerLysGluAlaTyrIleLysAspLeuLeu    610615620    LysTyrSerSerAspIleValVal    625630    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 57 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "SYNTHETIC OLIGONUCLEOTIDE"    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    TTTACAGAATTCCCCATGGTAAACATGGTTTCTCTGCGCGACAGAGACAAAAGTTAA57    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "synthetic oligonucleotide"    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    TAATTTGTCGGATCCCATGGGTAGACCTAGTTGCTCGAA39    __________________________________________________________________________

What is claimed is:
 1. An isolated and purified DNA molecule comprisingessentially of DNA encoding the NIa protease of the FLA.83 W-type strainof papaya ringspot virus.
 2. The isolated and purified DNA molecule ofclaim 1 consisting of DNA encoding the NIa protease of the FLA.83 W-typestrain of papaya ringspot virus.
 3. The isolated and purified DNAmolecule of claim 1 from the FLA.83 W-type strain of papaya ringspotvirus having the nucleotide sequence shown in SEQ ID NO:
 1. 4. A vectorcomprising a chimeric expression cassette comprising the DNA molecule ofclaim 1, a promoter and a polyadenylation signal, wherein the promoteris operably linked to the DNA molecule, and the DNA molecule is operablylinked to the polyadenylation signal.
 5. The vector of claim 4 whereinthe promoter is the cauliflower mosaic virus 35S promoter.
 6. The vectorof claim 5 wherein the polyadenylation signal is the polyadenylationsignal of the cauliflower mosaic 35S gene.
 7. A bacterial cellcomprising the vector of claim
 4. 8. The bacterial cell of claim 7wherein the bacterial cell is selected from the group consisting of anAgrobacterium tumefaciens cell and an Agrobacterium rhizogenes cell. 9.A transformed plant cell transformed with the vector of claim
 4. 10. Thetransformed plant cell of claim 9 wherein the promoter is cauliflowermosaic virus 35S promoter and the polyadenylation signal is thepolyadenylation signal of the cauliflower mosaic 35S gene.
 11. A plantselected from the family Cucurbitaceae comprising a plurality of thetransformed cells of claim
 10. 12. An isolated and purified DNA moleculecomprising DNA encoding the NIa protease of the USA P-type (HAattenuated) strain of papaya ringspot virus.
 13. The isolated andpurified DNA molecule of claim 12 consisting of DNA which encodes theNIa protease of the USA P-type (HA attenuated) strain of papaya ringspotvirus.
 14. The isolated and purified DNA molecule of claim 12 from theUSA P-type (HA attenuated) strain of papaya ringspot virus having thenucleotide sequence shown in SEQ ID NO:5.
 15. A vector comprising achimeric expression cassette comprising the DNA molecule of claim 12, apromoter and a polyadenylation signal, wherein the promoter is operablylinked to the DNA molecule, and the DNA molecule is operably linked tothe polyadenylation signal.
 16. The vector of claim 15 wherein thepromoter is the cauliflower mosaic virus 35S promoter.
 17. The vector ofclaim 15 wherein the polyadenylation signal is the polyadenylationsignal of the cauliflower mosaic 35S gene.
 18. A bacterial cellcomprising the vector of claim
 15. 19. The bacterial cell of claim 18wherein the bacterial cell is selected from the group consisting of anAgrobacterium tumefaciens cell and an Agrobacterium rhizogenes cell. 20.A transformed plant cell transformed with the vector of claim
 15. 21.The transformed plant cell of claim 20 wherein the promoter iscauliflower mosaic virus 35S promoter and the polyadenylation signal isthe polyadenylation signal of the cauliflower mosaic 35S gene.
 22. Aplant selected from the family Cucurbitaceae comprising a plurality ofthe transformed cells of claim
 21. 23. A method of preparing a papayaringspot viral resistant plant comprising:(a) transforming plant cellswith a chimeric expression cassette comprising a promoter functional inplant cells operably liked to a DNA molecule that encodes a protease;wherein the DNA molecule is obtained from a papaya ringspot virus strainselected from the group consisting of FLA.83 W-type and USA P-type (HAattenuated); (b) regenerating the plant cells to provide adifferentiated plant; and (c) identifying a transformed plant thatexpresses the papaya ringspot protease gene at a level sufficient torender the plant resistant to infection by papaya ringspot virus. 24.The method of claim 23 wherein the DNA molecule is obtained from apapaya ringspot virus strain having the nucleotide sequence show in SEQID NO: 1 or SEQ ID NO:
 5. 25. The method of claim 23 wherein the plantis a dicot.
 26. The method of claim 23 wherein the dicot is selectedfrom the family Cucurbitaceae.
 27. A vector comprising a chimericexpression cassette comprising the DNA molecule of claim 1 and at leastone chimeric expression cassette comprising a heterologous PRV proteasegene, a PRVcoat protein gene, a cucumber mosaic virus coat protein gene,a zuchini yellow mosiac virus coat protein gene, or a watermelon mosaicvirus-2 coat protein gene, wherein each expression cassette comprises apromoter and a polyadenylation signal wherein the promoter is operablylinked to the DNA molecule, and the DNA molecule is operably linked tothe polyadenylation signal.
 28. A bacterial cell comprising the vectorof claim
 27. 29. A transformed plant cell transformed with the vector ofclaim
 27. 30. The transformed plant cell of claim 29 wherein thepromoter is cauliflower mosaic virus 35S promoter and thepolyadenylation signal is the polyadenylation signal of the cauliflowermosaic 35S gene.
 31. A vector comprising a chimeric expression cassettecomprising the DNA molecule of claim 12 and at least one chimericexpression cassette comprising a heterologous PRV protease gene, aPRVcoat protein gene, a cucumber mosaic virus coat protein gene, azuchini yellow mosiac virus coat protein gene, or a watermelon mosaicvirus-2 coat protein gene, wherein each expression cassette comprises apromoter and a polyadenylation signal wherein the promoter is operablylinked to the DNA molecule, and the DNA molecule is operably linked tothe polyadenylation signal.
 32. A bacterial cell comprising the vectorof claim
 31. 33. A transformed plant cell transformed with the vector ofclaim
 31. 34. The transformed plant cell of claim 33 wherein thepromoter is cauliflower mosaic virus 35S promoter and thepolyadenylation signal is the polyadenylation signal of the cauliflowermosaic 35S gene.